The overall approach that we have taken in conducting this project has been to perform biochemical studies using model reactions that mimic as closely as possible natural reactions that occur during HIV replication. By focusing on the natural reaction, we demonstrated that a posttranscriptional modification in the priming tRNA lys3 is absolutely essential for an in vitro replication and presumably for infection. The defect is in the (+) strand strong stop and strand transfer stage of the reaction. Without the tRNA modification, replication does not terminate at the appropriate location and replication aborts after strand transfer, because the 3'-terminus is not complementary to the template. We will test the hypothesis that the critical modification is m1A58, the modified nucleotide just after the proper stop signal for synthesis of the (+) strand strong stop intermediate. This will be accomplished by modifying synthetic tRNA with tRNA (adenin-1)-methyltransferase and testing whether the resulting tRNA is sufficient to support HIV replication. We present plans to test the concept that human tRNA A58 adenine N-1 methytransferase by an antisense approach and by assembling HIV particles in vivo that contain mutant tRNAs that cannot be methylated at the 58 position. The infectivity of virus produced will be determined and compared with control infections. During the next grant period we will also focus on the factors required for formation of a specific nucleoprotein complex that initiates synchronous rapid synthesis and strand transfer in the (-) strand reaction. The mechanism of (-) strand transfer will be determined using natural HIV sequence templates.